Infectious diseases caused by existing and new emerging viruses pose a major threat to human and animal health, in both developed and developing nations. Although some degree of control is achieved by using vaccines and antiviral drugs, such approaches are only effective for a small proportion of these viruses. Recent advances in medicine and molecular biology have uncovered novel approaches and technologies that could be harnessed in the fight against infectious diseases. The discovery of RNA interference (RNAi) as an evolutionarily conserved and sequence-specific gene silencing mechanism in eukaryotes (Fire, A. et al. (1998) Nature 391, 806-11) has paved way for developing RNAi as a powerful therapeutic tool against pathogenic agents such as viruses.
RNA interference (RNAi) is a naturally occurring cellular mechanism of gene suppression that functions in both plants and animals. The conserved RNAi pathway involves the processing of double stranded RNA (dsRNA) duplexes into 21-23 nucleotide (nt) molecules, known as small interfering RNAs (siRNA), to initiate gene suppression (Hannon, (2002) RNA interference. Nature 418, 244-51). In mammalian systems, the cellular processing of long dsRNA can induce an interferon (IFN) mediated antiviral defence mechanism that ultimately leads to non-specific translational shutdown and apoptosis (Stark et al., (1998) Annu Rev Biochem 67, 227-64, Williams, (1997) Biochem Soc Trans 25, 509-13). However, this non-specific cellular activity can be circumvented by the direct transfection of in vitro synthesized siRNAs of up to 30 nucleotides (nt) in length (Elbashir et al., (2001) Nature 411, 494-8). Since this discovery, the development of DNA-based vectors for expression of short hairpin RNA (shRNA) molecules that are processed within the cell to produce active siRNA molecules has progressed rapidly (Brummelkamp et al., (2002) Science 296, 550-3, Yu et al., (2002) Proc Natl Acad Sci USA 99, 6047-52). Such shRNA expression vectors often feature promoters of a small class of RNA polymerase III (pol III) promoters such as U6 and 7SK. There has been a total of five separate U6 promoters described from the human genome (Domitrovich & Kunkel, Nucleic Acids Res 31, 2344-52 (2003), each loci displayed differential transcriptional activities, a finding which has also been seen for the chicken (Kudo & Sutou (2005) J Reprod Dev 51, 411-7, Wise et al., (2007) Anim Biotechnol 18, 153-62) and the cow (Lambeth et al., (2006) Anim Genet 37, 369-72).
While initial demonstrations of RNAi in mammalian cells showed suppression of cellular transcripts, more recently both siRNAs and shRNA have been shown to suppress replication of a number of viruses in vitro and in vivo. For example, the efficient inhibition of human pathogens such as hepatitis C virus (Randall & Rice, (2004) Virus Res 102, 19-25), human immunodeficiency virus-1 (Coburn & Cullen, (2002) J Virol 76, 9225-31) and influenza A (Ge et al., (2003) Proc Natl Acad Sci U S A 100, 2718-23), as well as livestock viruses such as foot and mouth disease virus (Chen et al., (2004) J Virol 78, 6900-7, Liu et al., (2005) Virology 336, 51-9) by RNAi have been described. The use of RNAi to inhibit the replication of several herpesviruses has also been reported, including murine herpesvirus 68 (Jia & Sun, (2003) J Virol 77, 3301-6), Epstein-Barr virus (Chang et al., (2004) Gen Virol 85, 1371-9) HSV-1 (Bhuyan et al., (2004) J Virol 78, 10276-81) herpesvirus-6B (Yoon et al., (2004) J Biochem Mol Biol 37, 383-5), human cytomegalovirus (Wiebusch et al., (2004) J Gen Virol 85, 179-84), Kaposi sarcoma-associated herpesvirus (Godfrey et al., (2005) Blood 105, 2510-8), duck herpesvirus (Mallanna et al., (2006) Virus Res 115, 192-7) and HSV-2 (Palliser et al., (2006) Nature 439, 89-94).
These studies have involved the introduction of synthetic siRNA, or the genetic transfer of DNA expression cassettes capable of producing siRNA or shRNA in human cells. While this approach may give valuable information about the mechanism of RNAi using cells in culture, it can be difficult to achieve the same effect in vivo. There are difficulties associated with expressing the siRNA in the target cell type and achieving sufficient and sustainable levels of expression.
Although the specific and effective nature of RNAi is potentially a highly powerful therapeutic tool, sustainable long-term target gene knockdown still remains a hurdle for widespread therapeutic use of RNAi. Delivery of siRNAs through the use of recombinant viral vectors such as adenoviruses, adeno-associated viruses (AAV) and lentiviruses can overcome some of these problems. However, concerns on the safety of some of these vectors and over-saturation of the RNAi pathway due to higher expression levels of siRNAs are to be tackled (Aagard and Rossi (2007) Adv. Drug Delivery Reviews 59, 75-86).
There is thus a need for an improved RNAi delivery mechanism giving sustainable long-term levels of expression of RNAi in vivo.